We are currently investigating DNA complexes of the type II restriction enzyme, EcoRV. Typically restriction endonucleases can distinguish between specific recognition and nonspecific DNA sequences quite efficiently in the absence of divalent metal co-factors that are required for cleavage. At present, however, results in literature suggest that EcoRV has unusually low sequence stringency. We have applied a self-cleavage assay, developed previously by us, to measure EcoRV-DNA competitive binding and to evaluate the influence of water activity, pH and salt concentration on the binding stringency of the enzyme in the absence of divalent ions. This technique monitors only enzymatically competent complexes of the endonuclease. It does not have the limitations of gel mobility shift assay while providing same level of sensitivity. We find the enzyme can readily distinguish specific and nonspecific sequences. The relative specific-nonspecific binding constant increases strongly with increasing neutral solute concentration and with decreasing pH. The difference in number of associated waters between specific and nonspecific DNA-EcoRV complexes is consistent with the differences in the crystal structures. Despite the large pH dependence of the sequence specificity, the osmotic pressure dependence indicates little change in structure with pH. Importantly, the large osmotic pressure dependence means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowed environment of the cell. In addition to divalent ions, water activity and pH are key parameters that strongly modulate binding specificity of EcoRV. We found that the EcoRV has quite unusual kinetics of specific complex formation in the absence of divalent ions that was not observed for EcoRI. A significant fraction of the total enzyme, 45%, forms enzymatically competent complexes unusually slowly, especially at pH 7.6. This novel result can be explained by a very slow transition between two conformations of the free enzyme in solution. The equilibrium distribution of the slowly and quickly associating protein structures and their exchange kinetics may depend on many parameters including pH, salt, osmolytes, and divalent cations. The slow rate of complex formation could explain the lack of specificity reported by others. The slow rate of EcoRV complex formation we observe necessarily dictates longer incubation times than is typical to reach equilibrium especially at pH values higher than 7.0. This may account for some of the difference in competitive binding constants. The observation of at least two kinetics components in association indicates that EcoRV is an allosteric protein with at least two conformations. Allosterism is now recognized as important concept for DNA-protein complexes, offering an additional level of control over binding and activity. The recognition specificity or activity of DNA binding proteins can be modulated by ligands or proteins that bind to one allosteric conformation in preference to others. We are continuing our investigation into the EcoRV structures responsible for the different kinetic classes of association. The association and dissociation kinetics of sequence specific DNA binding proteins are surprisingly complicated. In association, proteins initially bind nonspecifically and slide along the DNA to either find the specific sequence or dissociate and start the process again. Sliding allows the protein to scan a region of DNA. It also enables the protein to locate its recognition sequence faster than diffusion in solution would allow. If the recognition sites of transcription factors are normally occluded by nucleosomes, there may be a limited time period during which nucleosomes are transiently displaced for these factors to find their sites. The period between dissociation and subsequent association steps is termed the hopping or jumping time. The dissociation process is just the opposite;the specifically bound protein will transition to a nonspecifically bound form at the recognition site and start sliding along the DNA to either rebind to the specific site or dissociate into solution. We have uncovered a novel method to probe the hopping process. Dissociation kinetics can be measured by adding oligonucleotide containing the specific site to a solution of a longer DNA fragment with prebound protein. Protein that dissociates from the DNA fragment is trapped by the added competitor. We use a gel mobility shift assay variant or a self-cleavage assay that we have developed to measure the loss of fragment bound protein with time. The ratio of specific site concentrations of oligonucleotide and of DNA fragment is at least 100 and is usually higher. At these high ratios, if protein was added to the mixture of the two, the probability that the protein will bind to the DNA fragment is <1%. We find, however, that the dissociation kinetics of both EcoRI and EcoRV depend on the oligonucleotide concentration. We surmise that this dependence is due to protein hopping kinetics. After the initial dissociation of protein from DNA, it is still close to the DNA. The probability that the protein will rebind to the same DNA is quite large. Mathematical expressions are available for the distribution of times that a protein that dissociates at time 0 will rebind to the original DNA fragment at time t assuming no other DNA is around. During this off-time the protein can bind the oligonucleotide. The probability that a protein will be captured by an oligonucleotide during a hopping excursion can be calculated from the oligonucleotide concentration, the association rate constant, and the hopping time distribution function. A distribution function determined by random walk simulations gives a reasonably good description of the oligonucleotide concentration dependence observed for EcoRI. The analytical expression for 2-dimensional first passage times, however, predicts a much smaller dependence than is observed. We suspect that the electrostatic attraction between protein and DNA is the reason the analytic expression fails. Unlike EcoRI, the dependence of dissociation rate on oligonucleotide concentration for EcoRV is pH sensitive. At low pH values the oligonucleotide concentration dependence is about the same as for EcoRI. There is much less dependence at pH 7.5. Our working hypothesis is that a return to the DNA does not necessarily mean a return to the recognition site;there is a probability that the protein will dissociate again before reaching the recognition site again. In the limit of no probability of rebinding of the specific sequence there will be no oligonucleotide concentration dependence. That pH can affect this probability means that either pH affects the relative sliding and dissociation rates or that the protein can dissociate in a pH dependent conformation that is not able to rebind to the DNA. This latter possibility is attractive since the search process will be more efficient;the protein must diffuse further from the original binding region before being able to rebind to DNA. We have also further developed a method for stabilizing labile DNA-protein complexes for analysis by the gel mobility shift assay. We have shown that 30% triethylene glycol in the gel (equivalent to 4.3 osmolal) is enough to stabilize completely weak complexes that have dissociation constants at regular salt and pH conditions in the micromolar range. We are now further extending this approach to other techniques for separating complex and free components as gel chromatography and capillary electrophoresis.